U.S. patent application number 11/547675 was filed with the patent office on 2009-01-01 for method for modulating appetite.
This patent application is currently assigned to ST VINCENT'S HOSPITALSYDNEY LIMITED. Invention is credited to Samuel Norbert Breit.
Application Number | 20090004181 11/547675 |
Document ID | / |
Family ID | 35149777 |
Filed Date | 2009-01-01 |
United States Patent
Application |
20090004181 |
Kind Code |
A1 |
Breit; Samuel Norbert |
January 1, 2009 |
Method for Modulating Appetite
Abstract
The present invention provides a method of modulating appetite
and/or body weight in a subject, said method comprising
administering to said subject an effective amount of a MIC-1
modulating agent, wherein said agent increases or decreases the
amount of MIC-1 present in said subject, or inhibits or enhances
the biological activity of present in said subject.
Inventors: |
Breit; Samuel Norbert; (New
South Wales, AU) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
ST VINCENT'S HOSPITALSYDNEY
LIMITED
Sydney
AU
|
Family ID: |
35149777 |
Appl. No.: |
11/547675 |
Filed: |
April 13, 2005 |
PCT Filed: |
April 13, 2005 |
PCT NO: |
PCT/AU05/00525 |
371 Date: |
October 6, 2006 |
Current U.S.
Class: |
424/133.1 ;
424/130.1; 435/14 |
Current CPC
Class: |
A61P 3/04 20180101; A61P
1/14 20180101; G01N 2800/303 20130101; A61P 3/00 20180101; A61K
38/1709 20130101; C07K 2317/76 20130101; A61K 2039/505 20130101;
C07K 16/22 20130101; C07K 16/24 20130101; C07K 2317/24 20130101;
A61P 43/00 20180101; G01N 2800/02 20130101; A61K 38/19 20130101;
A61P 1/04 20180101 |
Class at
Publication: |
424/133.1 ;
424/130.1; 435/14 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 3/04 20060101 A61P003/04; C12Q 1/54 20060101
C12Q001/54 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 13, 2004 |
AU |
2004901957 |
Claims
1. A method of modulating appetite and/or body weight in a subject,
said method comprising administering to said subject an effective
amount of a MIC-1 modulating agent, wherein said agent increases or
decreases the amount of MIC-1 present in said subject, or inhibits
or enhances the biological activity of MIC-1 present in said
sample.
2. A method for increasing appetite and/or body weight in a
subject, said method comprising administering to said subject an
effective amount of a MIC-1 inhibiting agent optionally in
admixture with a pharmacologically-acceptable carrier and/or
excipient.
3. A method according to claim 2, wherein said MIC-1 inhibiting
agent is an agent which decreases the amount of endogenous MIC-1 in
said subject.
4. A method according to claim 3 wherein said agent is selected
from the group consisting of anti-MIC-1 antibodies or fragments
thereof, catalytic and inhibitory molecules targeted against the
MIC-1 gene and inhibitors of MIC-1 transcription or
translation.
5. A method according to claim 2, wherein said MIC-1 inhibiting
agent is an agent which inhibits the activity of endogenous MIC-1
in said subject.
6. A method according to claim 5, wherein said agent is selected
from the group consisting of anti-MIC-1 antibodies or fragments
thereof, soluble extra-cytoplasmic receptor domains of MIC-1
receptors, soluble molecules or matrix-associated proteins that
bind to MIC-1 and peptide, peptide mimetic or small organic
molecule inhibitors of MIC-1 binding to its receptor.
7. A method according to claim 1 wherein said MIC-1 inhibiting
agent is a humanised monoclonal anti-MIC-1 antibody.
8. A method according to claim 2, wherein the subject is suffering
from decreased appetite and/or weight loss associated with advanced
cancer.
9. A method for decreasing appetite and/or body weight in a
subject, said method comprising administering to said subject an
effective amount of a MIC-1 enhancing agent optionally in admixture
with a pharmacologically-acceptable carrier and/or excipient.
10. A method according to claim 9, wherein said MIC-1 enhancing
agent is an agent which enhances the amount of endogenous MIC-1 in
said subject.
11. A method according to claim 10, wherein said agent is selected
from the group consisting of MIC-1, agents which enhance
transcription or translation of the MIC-1 gene and agents which
enhance p53 expression or activity.
12. A method according to claim 9, wherein the subject is suffering
from obesity.
13. A method for assessing the effect of an agent on the appetite
and/or body weight of a subject, said method comprising
administering said agent to said subject or a suitable animal model
thereof and detecting any increase or decrease in the amount of
MIC-1 in said subject or animal model.
14. A method for assessing the effect of an agent on the appetite
and/or body weight of a subject, said method comprising forming a
mixture between MIC-1 or a functional fragment or mimetic thereof,
a MIC-1 binding partner, and said agent, and detecting any increase
or decrease in binding between the MIC-1 or a functional fragment
or mimetic thereof and the MIC-1 binding partner.
15. A method for assessing the effect of an agent on the appetite
and/or body weight of a subject, said method comprising exposing a
cell expressing MIC-1 to said agent and detecting any increase or
decrease in the level of said expression of MIC-1.
16. A method of assessing appetite in a subject, said method
comprising determining the amount of MIC-1 present in the serum in
said subject.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a method for modulating appetite
and/or body weight in an individual. In one particular application,
the method involves administering to a subject suffering from
decreased appetite and/or weight loss associated with late stage
tumours (particularly, cancers), an effective amount of an agent
which is capable of decreasing the amount of macrophage inhibitory
cytokine-1 (MIC-1) present in the subject or which otherwise
inhibits the activity of MIC-1 in the subject.
BACKGROUND TO THE INVENTION
[0002] The control of body weight is a complex process that at
present is incompletely understood. It is multifactorial and is
influenced by appetite, food ingestion and excretion, energy
utilisation and expenditure. A number of soluble mediators are
known to be involved in regulating various aspects of this process
and include hormones and cytokines such as leptin, ghrelin,
melanocortin, agouti-related peptide, and neuropeptide Y (NPY).
Normal weight control is important to good health and obesity
especially, may greatly increase morbidity and mortality in
individuals. Lower than average weight can also be problematic, and
in developed societies, where sufficient food is available, this is
more frequently due to diseases including some chronic inflammatory
disorders, eating disorders such as anorexia nervosa, and cancer.
Especially in the late stages of cancer, cachexia is common
(occurring in most terminally ill cancer patients), and is
responsible for about a quarter of all cancer-related deaths.
[0003] Some years ago, the present applicant cloned and
characterised a novel human TGF-.beta. superfamily cytokine that
was named macrophage inhibitory cytokine-1 (MIC-1) (1-7), but has
since also become known as prostate derived factor (PDF), placental
bone morphogenetic protein (PLAB), and growth/differentiation
factor-15 (GDF-15) (7). Under resting conditions, placenta is the
only tissue expressing large amounts of MIC-1 (7), but epithelial
cells in a wide variety of other organs also normally express small
amounts of MIC-1 mRNA. This low level of normal MIC-1 expression
is, however, dramatically increased in malignancy, inflammation and
injury (7, 8-11), the increase being induced by a wide variety of
cell stress and activation factors, and is mediated
intracellularly, particularly by the transcription factor p53 and
EGR-1 (12-15). In particular, increased MIC-1 expression has been
strongly linked to breast, prostate, pancreatic and colon cancers
(9-11, 17, 18), and in a recently published study (20) of several
hundred patients with colonic polyps or colon cancer, the present
applicant showed that elevation of serum levels of MIC-1 occurs in
a progressive stepwise manner, reflecting colon cancer
pathogenesis, with progression from normal to benign and then to
dysplastic colonic polyps and finally colon cancer. This
observation, along with results from other studies (15, 17, 19,
21), suggests that MIC-1 has an important role in tumour
progression, by inducing significant paracrine effects modulating
the tumour environment.
[0004] In work leading to the present invention, it had been
observed that the serum levels of MIC-1 of patients in the late
stages of one of the abovementioned epithelial cancers (eg serum
levels of 10 to 50 ng/ml or more), correlated with serum levels in
mice which were over-expressing MIC-1 and which showed marked
weight loss. It was therefore proposed that the cachexia commonly
exhibited in patients with cancer associated with increased MIC-1
expression, is due to the over-expression of MIC-1 and that by
inhibiting that expression (eg with anti-MIC-1 antibodies), it
would be possible to reverse or reduce the severity of the weight
loss.
SUMMARY OF THE INVENTION
[0005] Thus, in a first aspect, the present invention provides a
method of modulating appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-modulating agent, wherein said agent increases or
decreases the amount of MIC-1 present in said subject, or inhibits
or enhances the biological activity of MIC-1 present in said
subject.
[0006] In a second aspect, the present invention provides a method
for increasing appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-inhibiting agent optionally in admixture with a
pharmacologically-acceptable carrier and/or excipient.
[0007] In a third aspect, the present invention provides a method
for decreasing appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-enhancing agent optionally in admixture with a
pharmacologically-acceptable carrier and/or excipient.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 provides a schematic diagram of the processing of the
MIC-1 precursor through to its mature, 112 amino acid form.
Cleavage of the propeptide from the mature domain occurs at
Arg.sup.196.
[0009] FIG. 2 graphically shows the relationship between nude mouse
weight and human MIC-1 serum levels in blood collected when the
largest of the mouse tumours has reached about 1 cm diameter. Nude
mice were xenografted with human DU145 cells engineered to over
express either;
(i) full length human MIC-1 (including the propeptide) (series 3),
(ii) mature human MIC-1 (no propeptide) (series 1), (iii) human
MIC-1 including the propeptide but having the furin-like
proconvertase site deleted (FURIN DEL) (series 2), and (iv) vector
only negative control (series 4).
[0010] FIG. 3 graphically shows the relationship between nude mouse
percentage weight loss (compared to weight at the start of the
experiment) and human MIC-1 serum levels in blood collected when
the largest of the mouse tumours had reached about 1 cm diameter.
Nude mice were xenografted with human DU145 cells engineered to
over express;
(i) full length human MIC-1 (including the propeptide) (series 3),
(ii) mature human MIC-1 (no propeptide) (series 1), (iii) human
MIC-1 including the propeptide but having the furin-like
proconvertase site deleted (FURIN DEL) (series 2), and (iv) vector
only negative control (series 4).
[0011] FIG. 4 provides graphical results of the effect of sheep
antihuman MIC-1 antibodies on mouse weight (g). (A) On day 27, two
mice were given 10 mg (intraperitoneally) of purified IgG from
sheep immunised with highly purified recombinant MIC-1 to develop
high titre antibodies to human MIC-1. (B) On day 27, two mice were
give 10 mg (intraperitoneally) of control purified IgG from normal
sheep serum. The graphs A and B show representative data from one
of each of the mice in the two groups.
[0012] FIG. 5 provides the results of a weight loss assessment with
a MIC-1 over-expressing transgenic (TG) mouse line min 28. Body
weight was significantly reduced (P<0.001) in both male and
female min 28 mice compared to congenic wild type litter mates (3
litters, 59 to 61 days of age).
[0013] FIG. 6 provides the results of a weight loss assessment with
a MIC-1 over-expressing transgenic (TG) mouse line min 75. Body
weight was significantly reduced (P<0.001) in both male and
female min 75 mice compared to congenic wild type (WT) litter mates
(3 litters, 59 to 61 days of age).
[0014] FIG. 7 shows a comparison of body weight (g), of wild type
mice (filled symbols, WT) and heterozygous transgenic litter mate
mice (TG, open symbols) from seven litters. The number indicates
the average weight of heterozygous mice compared to their wild type
litter mates within each litter. Newborn WT and TG mice (less than
mice<48 h old) are not significantly different in
bodyweights.
[0015] FIG. 8 shows that administration of a monoclonal antibody
(MAb26) to human MIC-1 can reverse the weight loss in nude mice
xenografted with human DU145 cells which have been transduced to
over-express MIC-1 using a construct of mature human MIC-1 (no
propeptide). Mice injected with DU145 cells over expressing MIC-1
started to lose weight rapidly. Administration of a single
injection of MAb26, in amounts between 0.1 and 1 mg, at day 11,
caused in increase in weight, the magnitude of which, and the
duration of which, increased with increasing amounts of MAb26
(A-C). There was no effect of MAb26 on tumour growth (D-F).
Untreated mice (G) and mice treated with PBS buffer alone (H)
rapidly and continuously lost weight over the course of the
experiment. Weight (g) on the vertical axis.
[0016] FIG. 9 shows a comparison of food intake, daily over 3
successive days, in nude mice xenografted with human DU145 cells
which have been transduced to over-express MIC-1 using a construct
of mature human MIC-1 (no propeptide) and control mice receiving
DU145 cells transduced with a control construct.
[0017] FIG. 10 shows a comparison of fat pad and muscle weights in
nude mice xenografted with human DU145 cells which have been
transduced to over-express MIC-1 using a construct of mature human
MIC-1 (no propeptide) and control mice receiving DU145 cells
transduced with a control construct. MIC-1 bearing DU145 expression
tumours are represented by solid bars and the open bars represent
mice bearing control tumours. Statistical comparison was undertaken
using T test and the number of stars indicates increasing
statistical significance from p=0.003 to p<0.0001. There was a
marked decrease in the weight of body fat in inguinal fat,
epididimal fat and retroperitoneal fat. There was no significant
difference in the muscle weight between the two groups of mice.
NS=not significant **p<0.01 ***p<0.001.
[0018] FIG. 11 shows food intake in MIC-1 transgenic mice compared
to wild type controls. 5 wild type (WT) and 6 transgenic (TG) mice
were individually housed in cages, and left for 48 hours to adjust
to the single housing. Food placed in the hopper was weighed at
time point zero. Every 24 hours, food consumed was estimated by
subtracting the refusal and the spillage from the weight of the
food put into the hopper. Food intake was measured over four,
separate 24 hour periods. Food intake per mouse/day was
significantly greater in WT animals (p<0.03) (A). However, this
difference disappeared when the food intake was corrected for the
body weight of the mouse (B).
[0019] FIG. 12 shows the weights of organs from MIC-1 transgenic
(TG) mice and wild type (WT) mice. Abbreviations: m=male, f=female,
epid=epidydimal, ut=uterine, retroperit=retroperitoneal.
**p<0.01 ***p<0.001.
[0020] FIG. 13 shows the results of assays for MIC-1 binding to
fetuin. Purified recombinant MIC-1 (in 0.1% BSA) was incubated with
fetuin-coated agarose beads. The beads were then washed and bound
material analysed by SDS-PAGE followed by Western blotting with
anti-MIC-1 antibody. Lane 1, purified recombinant MIC-1; lane 2,
MIC-1 bound to fetuin beads; lane 3, fetuin beads only; lane 4,
MIC-1 incubated with agarose beads only. The arrow indicates the
MIC-1 bands.
[0021] FIG. 14 sections of normal adult mouse brain in the region
of the hypothalamus and the third ventricle (V3) were cut an
subjected to (A) in situ hybridisation for MIC-1 using
.sup.35S-labelled RNA probe and autoradiography and (B)
immunohistochemistry using in house affinity purified polyclonal
antibodies to recombinant murine MIC-1. The sections show
expression of MIC-1 mRNA and proteins in the region of the arcuate
nucleus (.DELTA.N) and paraventlicular region.
DETAILED DESCRIPTION OF THE INVENTION
[0022] It had been previously found that many cancers, especially
of epithelial origin, over-express MIC-1 and that serum MIC-1
levels rise in patients with these cancers in proportion to the
stage and extent of the disease. Especially in late stages of
cancer, these serum levels can reach 10 to 50 ng/ml or more, levels
which in mice are associated with marked weight loss. By reducing
MIC-1 levels or the activity of MIC-1 in cancer patients, it is
expected that weight loss, and the subsequent ill-effects on
patient well-being and esteem, may be reversed or reduced. In turn,
this may assist in the patient's capacity to be treated for cancer
and positively respond to the therapy, and thereby reduce morbidity
and mortality.
[0023] Thus, in a first aspect, the present invention provides a
method of modulating appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-modulating agent, wherein said agent increases or
decreases the amount of MIC-1 present in said subject, or inhibits
or enhances the biological activity of MIC-1 present in said
subject.
[0024] Where the method is operated to decrease the amount of MIC-1
present in the subject (particularly, to decrease the serum level
of MIC-1), or inhibit the activity of MIC-1, the method may
increase appetite and/or lead to an increase in body weight or, at
least, a reduction in any loss of body weight in the subject. On
the other hand, where the method is operated to increase the amount
of MIC-1 (particularly, to increase the serum level of MIC-1), or
enhance the activity of MIC-1, the method may decrease appetite
and/or lead to a decrease in body weight or, at least, a reduction
in any gain of body weight in the subject.
[0025] In a second aspect, the present invention provides a method
for increasing appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-inhibiting agent optionally in admixture with a
pharmacologically-acceptable carrier and/or excipient.
[0026] The method of the second aspect involves the administration
of a MIC-1-inhibiting agent. Such an agent may decrease the amount
of endogenous MIC-1 in the subject (particularly, the serum level
of endogenous MIC-1), and may be selected from anti-MIC-1
antibodies or fragments thereof (eg Fab fragments or recombinant
scFv fragments (22)), catalytic and inhibitory oligonucleotide
molecules targeted against the MIC-1 gene (eg ribozymes, DNAzymes,
antisense RNA, and small inhibitory RNA (siRNA)), and inhibitors of
MIC-1 transcription or translation. Alternatively, the
MIC-1-inhibiting agent may inhibit the activity of endogenous MIC-1
in the subject, and may be selected from anti-MIC-1 antibodies or
fragments thereof (eg Fab fragments or recombinant scFv fragments),
soluble extra-cytoplasmic receptor domains of MIC-1 receptors,
other soluble molecules or matrix-associated proteins that bind to
MIC-1 (eg heparin, heparan sulphate and fetuin), and peptide,
peptide mimetic, or small organic molecule inhibitors of, for
example, MIC-1 binding to its receptor. Additionally, peptide,
peptide mimetic, or small organic molecule inhibitors might inhibit
the activity of endogenous MIC-1 by inhibiting MIC-1 receptor
phosphorylation, or transmission of signalling information from the
MIC-1 receptor to the cell nucleus, or action of the relevant
transcription factor(s) on the cell genome. Further, the
MIC-1-inhibiting agent may be an inhibitor of the proconvertase
enzyme responsible for cleaving the propeptide from the mature
MIC-1 protein domain. As is shown in example 1 hereinafter,
immature MIC-1 (ie proMIC-1) associates with the extracellular
matrix, and thus by inhibiting the proconvertase enzyme responsible
for processing of MIC-1, MIC-1 can be "locked up" in the
extracellular matrix. Proconvertase enzyme may be inhibited by, for
example, (a) transfection of cells with an alpha-1-antitrypsin
mutant, alpha-1-antitrypsin Portland, (b) polyarginine peptides;
and (c) peptides based on the sequence of the target protein for
the proconvertase, spanning the propeptide sequence and
proconvertase sequence of the target protein.
[0027] Preferably, the MIC-1-inhibiting agent is an anti-MIC-1
antibody or fragment thereof, and more preferably, a humanised
monoclonal anti-MIC-1 antibody. Humanised anti-MIC-1 antibodies may
be produced in accordance with the methods described in U.S. Pat.
No. 5,225,539 (the entire disclosure of which is incorporated
herein by reference).
[0028] The method of the second aspect is useful for the treatment
of a subject suffering from decreased appetite and/or weight loss
associated with inflammatory disease (eg rheumatoid arthritis)
and/or cancer (particularly, an epithelial cancer such as breast,
prostate, colonic, rectal, bladder and pancreatic cancer). The
method, however, may also be useful for the treatment of decreased
appetite and/or weight loss associated with any other disease,
condition or treatment wherein MIC-1 is over-expressed (eg injury,
inflammation, stress, and radiotherapy and chemotherapy). Subjects
suitable for treatment with the method of the second aspect may be
restricted to those showing MIC-1 over-expression or, at least, a
serum level of MIC-1 consistently at the high end of the normal
serum level of 200-1200 pg/ml. Such subjects can be selected by
detection of a high serum MIC-1 level (eg from a whole blood or
serum sample), using an assay for MIC-1 (eg a MIC-1 ELISA (4)).
[0029] Preferably, the method of the second aspect is used for the
treatment of a subject suffering from decreased appetite and/or
weight loss associated with advanced cancer, where a high total
tumour mass often leads to a high serum level of MIC-1.
[0030] In a third aspect, the present invention provides a method
for decreasing appetite and/or body weight in a subject, said
method comprising administering to said subject an effective amount
of a MIC-1-enhancing agent optionally in admixture with a
pharmacologically-acceptable carrier and/or excipient.
[0031] The method of the third aspect involves the administration
of a MIC-1-enhancing agent. Such an agent may increase the amount
of endogenous MIC-1 in a subject (particularly, the serum level of
endogenous MIC-1), and may be selected from MIC-1, and agents which
enhance transcription or translation of the MIC-1 gene (eg the p53
transcription factor, which is often seen in elevated levels in
diseases associated with MIC-1 over-expression, or agents which
enhance p53 expression or activity such as nutlin (23)).
Alternatively, the MIC-1-enhancing agent may enhance the activity
of endogenous MIC-1 in the subject. As used herein, the term
MIC-1-enhancing agent is to be regarded as including agents which
mimic the activity of MIC-1 (eg active MIC-1 fragments, peptide
mimetics of the active domains of MIC-1, and small organic
molecules which mimic MIC-1 activity).
[0032] The method of the third aspect is useful for the treatment
of a subject suffering from obesity or who might otherwise desire
weight loss for reasons of well-being or vanity.
[0033] MIC-1-modulating agents for use in the methods of the
present invention may be formulated into any suitable
pharmaceutical/veterinary composition or dosage form (eg
compositions for oral, buccal, nasal, intramuscular and intravenous
administration). Typically, such a composition will be administered
to the subject in an amount which is effective to modulate appetite
and/or body weight, and may therefore provide between about 0.01
and about 100 .mu.g/kg body weight per day of the MIC-1-modulating
agent, and more preferably providing from 0.05 and 25 .mu.g/kg body
weight per day of the MIC-1-modulating agent. A suitable
composition may be intended for single daily administration,
multiple daily administration, or controlled or sustained release,
as needed to achieve the most effective results.
[0034] In addition to the MIC-1-modulating agents identified above,
other MIC-1-modulating agents may be identified by screening
candidate agents or libraries of agents for an effect on the amount
of MIC-1 present in a subject and/or on the activity of MIC-1. In a
similar manner, agents for treatment of various diseases or
conditions could be assessed for undesirable side-effects on
appetite and/or body weight (eg undesirable suppression or
enhancement of appetite).
[0035] Thus, in a further aspect, the present invention provides a
method for assessing the effect of an agent on the appetite and/or
body weight of a subject, said method comprising administering said
agent to said subject or a suitable animal model thereof (eg a
mouse), and detecting any increase or decrease in the amount of
MIC-1 (particularly, the serum MIC-1 level) in said subject or
animal model.
[0036] Any increase or decrease in the amount of MIC-1 in the
subject or animal model may be identified by obtaining MIC-1
samples (eg whole blood or serum samples) before and after the
administration of said agent, and determining the respective amount
of MIC-1 in said samples (eg with a MIC-1 ELISA).
[0037] In a similar aspect, the present invention provides a method
for assessing the effect of an agent on the appetite and/or body
weight of a subject, said method comprising forming a mixture
between MIC-1 (or a functional fragment or mimetic thereof), a
MIC-1 binding partner (preferably, a MIC-1 receptor or functional
fragment or mimetic thereof), and said agent, and detecting any
increase or decrease in binding between the MIC-1 (or functional
fragment or mimetic thereof) and the MIC-1 binding partner.
[0038] An increase in binding may indicate that the agent is likely
to decrease appetite and/or body weight of a subject. On the other
hand, a decrease in binding may indicate that the agent is likely
to increase appetite and/or body weight of a subject.
[0039] Also, in a further similar aspect, the present invention
provides a method for assessing the effect of an agent on the
appetite and/or body weight of a subject, said method comprising
exposing a cell expressing MIC-1 to said agent and detecting any
increase or decrease in the level of said expression of MIC-1.
[0040] An increase in expression of MIC-1 may indicate that the
agent is likely to decrease appetite and/or body weight of a
subject. On the other hand, a decrease in expression of MIC-1 may
indicate that the agent is likely to increase appetite and/or body
weight of a subject.
[0041] Preferably, this method is conducted in vitro using a MIC-1
expressing cell or cell line selected from macrophages, epithelial
cells, endothelial cells and cell lines thereof.
[0042] In a still further aspect, the present invention provides a
method of assessing appetite in a subject, said method comprising
determining the amount of MIC-1 (particularly, the serum MIC-1
level) present in said subject.
[0043] Such a method may also be predictive of future body
mass.
[0044] The finding that MIC-1 over-expression appears to decrease
appetite and/or body weight in a subject, suggests that methods of
gene therapy to increase the level of MIC-1 in a subject may
provide an effective treatment of obesity. Therefore, the present
invention also contemplates gene therapy methods, and gene therapy
agents, for decreasing appetite and/or body weight in a subject,
comprising recombinant MIC-1 genes to bring about increased
endogenous MIC-1 expression. Vectors suitable for the introduction
of MIC-1 genes include recombinant adenoviral or
adenoviral-associated vectors, recombinant retroviral vectors,
recombinant lentivirus vectors, liposomes including linear DNA, and
transduced or transformed stem cells.
[0045] In order that the nature of the present invention may be
more clearly understood, preferred forms thereof will now be
described with reference to the following non-limiting
examples.
EXAMPLE 1
Regulation of Serum MIC-1 Levels
[0046] MIC-1, like other members of the TGF-.beta. superfamily of
proteins, is synthesised as a precursor containing an N-terminal
propeptide and a C-terminal mature MIC-1 domain. The precursor
undergoes disulphide-linked dimerisation in the endoplasmic
reticulum (ER) and, once dimerised, leaves the ER for the Golgi
apparatus, where a furin-like convertase cleaves it at a conserved
RXXR site (amino acid 196) (SEQ ID No: 1). This cleavage separates
the propeptide from the mature C-terminal domain and MIC-1 is thus
released as a 24.5 kD disulphide linked dimer (1)(FIG. 1).
[0047] It has been previously found that substantial amounts of
MIC-1 are normally secreted in an unprocessed form. For example, it
has been found that endogenous unprocessed proMIC-1 is secreted
from a variety of cells including the trophoblast cell line BeWo
(4), the prostate cancer cell lines LnCAP and PC3, the pancreatic
cell line Panc 1 and the monocytoid cell line U937. In the prostate
adenocarcinoma line, LnCAP, it has been found that unprocessed
proMIC-1 associates with the extracellular matrix (ECM), whilst
mature MIC-1 locates to the conditioned medium (24). Preliminary
studies with MDCK transfectants has also demonstrated that ECM
association is also mediated by a C-terminal region of the
propeptide at amino acids 144-195. Additionally, both purified
recombinant propeptide and proMIC-1 interact with heparin through
the same C-terminal region of the propeptide.
[0048] The association of proMIC-1 with the ECM, suggests that ECM
association may provide local storage of latent MIC-1, wherein
processing of the stored proMIC-1 would result in the rapid release
of mature MIC-1 (which has little affinity for ECM) into the
circulation. To test this concept, a tumour xenograft model in nude
mice (16) was developed.
Materials and Methods
[0049] Using the DU145 human prostate carcinoma line (17), which
makes no endogenous MIC-1 (largely because the cells produce no
functional p53) and is therefore useful as a vehicle for expressing
various human MIC-1 constructs, permanently transfected and
subcloned DU145 cell lines were generated which were transduced
with eukaryotic expression vectors (IRES II EGFP vector, Clontech)
containing sequences encoding either;
(i) full length human proMIC-1 (except using an FSH leader peptide,
rather than the natural leader)(1), (ii) mature human MIC-1 (no
propeptide, but including an FSH leader), (iii) human proMIC-1
(including an FSH leader) with a deletion of the amino acid
sequence RGRRRAR (SEQ ID No:2) including the furin-like
proconvertase site (shown in bold), thereby preventing processing
and subsequent release of mature MIC-1 from the propeptide, and
(iv) vector only negative control (5).
[0050] High expressing subclones were selected based on EGFP
expression. These cells were injected subcutaneously into the flank
of immunodeficient BALB/c nu/nu nude mice. Mice were monitored
regularly and their weight determined on a 2-3 daily basis. Mice
were sacrificed about 2 months after injection or when tumour
diameter reached 1.1 cm. Serum was obtained from these mice just
prior to sacrifice, for estimation of the level of human MIC-1 by
ELISA (4, 16, 18). This ELISA for human MIC-1 does not cross react
with murine MIC-1, and has been previously used for the successful
and exclusive measurement of human tumour MIC-1 levels in mice
(16).
Results
[0051] The results are shown in FIGS. 2 and 3. Only tumour mice
expressing mature MIC-1 showed a dramatically elevated level of
serum MIC-1. Mouse tumours expressing the FURIN DEL mutant of
MIC-1, which could not be processed normally and thus contained the
propeptide, had markedly lower serum MIC-1 levels. By extrapolation
from in vitro data, it appears that this result is due to tight
association of the FURIN DEL mutant with the ECM.
Discussion
[0052] The results obtained in this example indicate that the MIC-1
propeptide is important in regulating the distribution of MIC-1
between tissues and blood. As such, any substances that bind to the
MIC-1 propeptide (eg heparin and heparan sulphate), or otherwise
compete with matrix binding sites on the propeptide (eg recombinant
purified propeptide itself) would be expected to increase the level
of MIC-1 in the circulation. As a consequence, functions mediated
by serum MIC-1, including appetite, would be modulated.
EXAMPLE 2
Modulation of Appetite by MIC-1
[0053] Over the course of the investigation described in example 1,
it was noted that of the xenograft model mice, those bearing a
tumour over-expressing MIC-1, either lost weight, or did not gain
as much weight as control mice. Studies were therefore conducted to
determine the extent and reason for the observed effect on mice
weight.
Materials and Methods
[0054] The mice were weighed just before sacrifice and weight/%
weight loss compared against the measured serum MIC-1 levels (ie as
determined by ELISA described in example 1).
[0055] To assess whether serum MIC-1 levels were responsible for
observed weight loss, a second study was conducted wherein nude
mice were injected subcutaneously with the DU145 clone over
expressing mature human MIC-1 (and which we had previously
associated with the highest serum MIC-1 levels) and at day 27,
after the mice had lost substantial weight, injected
intraperitoneally with either 1 mg or 10 mg of control purified
sheep IgG or IgG purified from serum from sheep that had been
immunised with recombinant human MIC-1 and had high titre
antibodies to human MIC-1. This sheep anti-human MIC-1 IgG reacted
with high affinity to human MIC-1 and had been previously used in a
MIC-1 ELISA.
[0056] To further demonstrate that the observed weight loss was
mediated by MIC-1 and not another tumour-derived product, an
evaluation of weight loss was made of two transgenic mouse lines
(min 28 and min 75; both created in C57B16 mice) which over express
murine MIC-1 under the control of the macrophage specific c-fms
promoter.
Results
[0057] In the studies conducted with sheep anti-human MIC-1 IgG, it
was found that 1 mg of sheep anti-human MIC-1 IgG made no
difference to the weight of the mouse (data not shown), however 10
mg of anti-MIC-1 IgG (see FIG. 4A) induced a rapid weight gain in
the respective tumour bearing nude mice (cf the results shown in
FIG. 4B with 10 mg of control IgG). This weight gain peaked 5 to 6
days after administration of the antibodies, and then gradually the
mice began to lose weight over the following 7 to 10 days.
[0058] The results of the weight loss assessment in the transgenic
mice lines min 28 and min 75 are shown in FIGS. 5 to 7 and indicate
that these mice are also substantially smaller than their wild type
congenic littermates. In these mice, weight at birth is equal and
differences in weight start appearing after the first few weeks of
life.
Discussion
[0059] The observed weight loss was very dramatic in some mice and
was found to be related to the serum level of tumour-derived human
MIC-1. The mice transduced with a DU145 clone over expressing
mature human MIC-1 had by far the highest levels of serum MIC-1 and
these mice lost weight at a dramatic rate. Observation of animal
behaviour, indicated that a major reason for this, was a dramatic
reduction in food ingestion by these mice. The finding that the
weight loss could be reversed by administration with sheep
anti-MIC-1 IgG (but not control IgG) demonstrates that the weight
loss was due to MIC-1. This was corroborated by the weight loss
assessment with the transgenic mice lines min 28 and min 75. In
these mice, which have markedly elevated serum MIC-1 levels even
though MIC-1 expression is macrophage-specific, a significant
weight differential was observed as compared to congenic wild type
mice. This weight loss effect occurred after birth, since both the
transgenic mice lines and their congenic wild type litter mates had
identical birth weights (ie as measured 24 hours after birth).
EXAMPLE 3
Weight Loss Associated with MIC-1 Secreting Tumour is Reversed by
Administration of an Anti-MIC-1 Monoclonal Antibody
Results and Discussion
[0060] A xenograft model was established in nude mice (as described
above) into whose flanks were injected either DU145 cells
engineered to over-express mature MIC-1. Mice injected with DU145
cells over expressing MIC-1 started to lose weight rapidly.
Administration of a single injection of a monoclonal antibody to
MIC-1 (MAb26), in amounts between 0.1 and 1 mg, at day 11, caused
an increase in weight, the magnitude of which, and the duration of
which increased with increasing amounts of MAb26 (FIGS. 8A-C). At
the highest dose of approximately 1 mg, the weight had risen to the
pre-xenograft level and took approximately 17 days to decrease
again to the same weight as when the antibody was first
administered. There was no effect of MAb26 on tumour growth (FIGS.
8D-F) and untreated mice (FIG. 8G) and mice treated with phosphate
buffered saline (FIG. 8H) (PBS) alone, rapidly and continuously
lost weight over the duration of the experiment.
EXAMPLE 4
Effect on Food Intake in Mouse Xenograft Model
Materials and Methods
[0061] A xenograft model was established in nude mice (as described
above) into whose flanks were injected either DU145 cells
engineered to over-express mature MIC-1, or bear a control plasmid.
On day 8 after injection of the DU145 cells over-expressing MIC-1,
when the average tumour volume was 56 mm.sup.3 and the average
weight loss 7%, food intake was measured for 3 consecutive 24 hour
time periods. The mice were left in groups of 5 per cage. Food
placed into the hopper and litter were weighed at time point 0.
After 24 hours, food consumed was estimated by subtracting refusal
and spillage from food put into the hopper. Food intake for the
control mice was measured in the same way, but on day 21 after
tumour injection when the tumour volume had reached an average of
70 mm.sup.3.
Results
[0062] Mice injected with DU145 over-expressing MIC-1 ate
significantly less food (about 30%) on day 1, 2 and 3 (p=0.01,
0.0001 and 0.02) than the control mice (FIG. 9). A direct
measurement of fat mass in these mice indicated that MIC-1
over-expression was associated with a marked reduction in fat mass
in the epididymal, inguinal, and retroperitoneal areas with no
reduction in mass in two representative muscles (FIG. 10).
EXAMPLE 5
Measurement of Serum Metabolic Markers in Mouse Xenograft Model
Materials and Methods
[0063] A xenograft model was established in nude mice (as described
previously) into whose flanks were injected either DU145 cells
engineered to over-express MIC-1 or control DU145 cells. At 11-16
days after injection of the DU145 tumour cells over-expressing
MIC-1 and 21-30 days after injection of the control tumour, when
tumour volumes had reached 100-200 mm.sup.3, and, or the mice had
lost approximately 18% body weight, the mice were sacrificed. From
previous experiments it is known that serum levels of tumour
derived human MIC-1 are between 15 and 58 ng/ml. Serum was
collected by cardiac puncture and assayed for the metabolic markers
using commercial immunoassays. Statistical comparison was
undertaken using the student T test.
Results and Discussion
[0064] Measurement of a range of metabolic markers in mice
demonstrated a statistically significant reduction in MIC-1
over-expressing tumor mice of triglyceride and free fatty acids as
well as glucagon and IGF-1 (data not shown). There was also a
reduction in leptin levels that is consistent with reduction in fat
mass, an indication that it is very unlikely that MIC-1 reduced
food intake is mediated by MIC-1 stimulation of leptin. The
difference for glucose was just short of statistical significance
at p=0.053. These finding are largely in keeping with starvation
and loss of fat mass.
EXAMPLE 6
Measurement of Fat Pad and Muscle Weight in Mouse Xenograft
Model
Materials and Methods
[0065] A xenograft model was established in male nude mice (as
described above). Into the flanks of 20 mice were injected DU145
cells engineered to over-express MIC-1 and into 20 mice were
injected DU145 cells transduced with a control plasmid. At 11-16
days after injection of the DU145 tumour cells over-expressing
MIC-1 and 21-30 days after injection of the control tumour, when
tumour volumes had reached 100-200 mm.sup.3, and, or the mice had
lost approximately 18% body weight, the mice were sacrificed.
Interscapular brown adipose tissue, inguinal, epididymal, and
retroperitoneal fat and also tibialis and gastrocnemius muscle
carefully dissected, removed and weighed and the weight was
corrected for body weight.
Results and Discussion
[0066] There was no reduction in brown fat but there was a marked
decrease in the weight of body fat in inguinal fat, epididymal fat
and retroperitoneal fat (FIG. 10). There was no significant
difference in the muscle weight between the two groups of mice
(FIG. 10). However, using more sensitive total lean body mass
analysis using the PIXImus imager (GE Lunar) indicated that there
was an overall reduction in lean body mass. It also confirmed a
much greater reduction in total fat mass and abdominal fat
mass.
EXAMPLE 7
MIC-1 Transgenic Mice
Results and Discussion
[0067] Transgenic mice were engineered to over-express MIC-1 from
monocytoid cells under the control of the c-fms promoter. These
mice have systemically elevated MIC-1 levels, appear well and breed
normally. They are indistinguishable from wild type mice but do
show a significant growth retardation starting at about 3 weeks and
into adulthood (FIG. 5-7). This effect was observed in two
independent transgenic lines called min 75 and min 28.
[0068] Like the tumour xenograft mice, the MIC-1 over-expressing
transgenic mice ate significantly less than their wild type
counterparts, but this difference disappears if the food intake is
corrected for mouse weight (FIG. 11). It is believed that increased
MIC-1 levels from birth result in decreased food intake which
results in decreased size and the reach an equilibrium in which
their size is appropriate for their reduced food intake.
Measurement of the same metabolic markers in the transgenic
animals, as in the tumour xenografted mice only showed a
significant difference in IGF-1 levels, which are reduced in the
MIC-1 transgenic mice.
[0069] Measurement of fat mass in inguinal, epididymal/uterine and
retroperitoneal areas shows a decreased fat mass in the over
expressing transgenic mice that is more prominent in female
compared to male mice (FIG. 12). Beside a smaller spleen and a
larger thymus, all three analysed fat pads were reduced in size. In
absolute terms, there was no difference between the weights of WT
versus TG thymus.
EXAMPLE 8
Control of Serum MIC-1 Levels by Fetuin
[0070] The presence of serum MIC-1, at a mean concentration of 450
pg/ml in all individuals, suggests that like some other TGF-.beta.
superfamily cytokines, MIC-1 may bind to one or more circulating
modulators. The glycoprotein, fetuin is widely expressed in cells
and tissues and is present in blood serum. The following
investigation was made to determine whether MIC-1 may interact with
this glycoprotein.
Materials and Methods
[0071] Purified recombinant, mature MIC-1 (in 0.1% BSA) was
incubated with fetuin-coated agarose beads. The beads were then
washed and bound material analysed by SDS-PAGE followed by Western
blotting with anti-MIC-1 antibody: Lane 1, purified recombinant
MIC-1; Lane 2, MIC-1 bound to fetuin beads; Lane 3, fetuin beads
only; Lane 4, MIC-1 incubated with agarose beads only.
Results
[0072] The results, shown in FIG. 13, clearly indicate that mature
MIC-1 interacts and binds with fetuin.
Discussion
[0073] As MIC-1 binds to fetuin, fetuin may offer an alternative to
the administration of anti-MIC-1 antibodies for modulating
functions mediated by serum MIC-1. For example, for modulating the
inhibitory effects of serum MIC-1 on appetite, fetuin could be
administered to a subject (eg a subject suffering from advanced
cancer) by a suitable route (eg intravenous administration) so as
to reduce the level of "free" MIC-1.
EXAMPLE 9
Analysis of MIC-1 Expression in Normal Mouse Brain
Results and Discussion
[0074] Food intake and appetite are controlled by a complex array
of mechanisms, many of which are located within the central nervous
system. The area within the nervous system controlling many basal
bodily functions such a appetite and body temperature are localised
within the area of the hypothalamus. In the case of appetite, many
of the complex factors regulating this process are localised to the
arcuate nucleus of the hypothalamus and many of the mediators and
receptors for mediators such as neuropeptide Y are localised in
this area. The blood brain barrier in this area is also leaky and
it is one of the very limited areas of the brain where there is an
opportunity for systemic molecules to cross the blood brain barrier
and act directly in the brain. It is considered that MIC-1 is able
to exert a direct effect on the acuate nucleus and hypothalamus by
this mechanism. However. MIC-1 is also expressed within this region
of the normal mouse brain (FIG. 14). It does not represent
diffusion of circulating MIC-1 as indicated by studies of in situ
bybridisation which demonstrate co-localisation of MIC-1 mRNA and
protein in the area of the acuate nucleus, periventricular area and
paraventricular hypothalamus. The localisation of MIC-1 in those
areas of normal brain, strongly associated with functions such as
appetite control, provides a strong argument for the role of MIC-1,
both from the peripheral circulation, and endogenously produced
within the brain, in controlling this important function.
Throughout this specification the word "comprise", or variations
such as "comprises" or "comprising", will be understood to imply
the inclusion of a stated element, integer or step, or group of
elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or
steps.
[0075] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context for the present invention. It is not to be taken as an
admission that any or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present invention as it existed in Australia or elsewhere
before the priority date of each claim of this application.
[0076] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
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Sequence CWU 1
1
214PRTArtificial Sequenceconsensus sequence for proconvertase 1Arg
Xaa Xaa Arg127PRTArtificial Sequencesequence including furin-like
proconvertase site 2Arg Gly Arg Arg Arg Ala Arg1 5
* * * * *